A previously known method for detecting a protein comprises steps of allowing an antibody specifically binding to a protein immobilized on a carrier for immobilization such as a membrane, and being radiolabeled with a radioisotope, to react with the protein; removing an unreacted labeled antibody portion by washing; and detecting the residual radiolabel, so as to detect the protein.
Such a detection method using a radiolabeled antibody is useful in that it allows for high sensitivity. However, it requires special equipment for treating radioactive compounds, and thus, it is hard to deal with. Moreover, the method is also problematic regarding disposal of radioactive substances. Furthermore, since a radioactive substance has a half-life, a radiolabeled antibody can be used only for a certain period of time after production thereof. Further, when the amount of DNA to be detected is small, this detection method requires a long period of time (several days to several weeks) as an exposure time for the autoradiography. In order to solve these problems, a nonradioactive detection method has been developed.
For example, there has been known a method of using an antibody into which a biotin molecule is introduced, wherein the biotin molecule of the antibody which was bound to a target substance is detected by using a (strepto)avidin-labeled enzyme complex. A protein can be detected with relatively high sensitivity in this detection system via biotin/(strepto)avidin. However, this method has disadvantages in that biotin, which is vitamin, is often generated in a biological sample, and the interaction of biotin/(strepto)avidin is thereby likely to be prevented during detection.
Japanese Patent Laid-Open (Kokai) No. 1-215300 describes a detection system using digoxigenin (DIG). In this detection system, digoxigenin is used as a label binding to an antibody. To detect the label, an anti-digoxigenin antibody to which alkaline phosphatase was bound is allowed to bind to digoxigenin, and thereafter, the label is detected by color development of a substrate as a result of the catalytic reaction of the alkaline phosphatase.
In the aforementioned detection system using DIG however, an antibody against a target substance is labeled with DIG However, a DIG derivative has disadvantages in that it is poor in water solubility and it is difficult to achieve efficient labeling of an antibody therewith.